Isolation of bioactive compounds from low-cost agricultural resources and its utilization in daily life

Abstract The ethanolic (80 %), methanolic (80 %) and aqueous decoction (100 % distilled water) of whole plant of Oxalis corniculata Linn (Indian Sorrel) was evaluated for its anti‐microbial and antioxidant properties by in vitro methods. Methanolic (80 %) and ethanolic (80 %) decoctions showed significant antibacterial property against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Salmonella typhi bacterial strains. In comparison to Chloramphenicol (C30) against bacteria, 80 % ethanolic decoctions showed significant effect, among the decoctions. Nowadays though the standard soap is in huge demand but it’s also facing major backlash due to the presence of synthetic compounds in it, which over long use may cause harmful effects on the skin health. Therefore, the organic soaps which are made up of natural ingredients, herbs or any sort Ayurvedic compound have fewer side effects on the human skin and are much safer than standard daily soap. The formulated therapeutic soap exhibits a significant amount of reducing potential (high FRAP and TAC values) and antioxidant activity (DPPH, ABTS assay).


InTRoDuCTIon
Bioactive compounds are referred to as the extra nutritional food components present in small amounts in plant-based foods that governs diverse health benefits [1].In plants, the bioactive compounds are mainly produced in the form of secondary metabolites [2].Various disorders are treated locally with Indian Sorrel.It is used as an antibacterial, cooling agent, niacin, vitamin C, and β-carotene.It also functions as an adjunctive therapy for the treatment of wounds [3][4][5].Avoiding the use of risky synthetic chemicals in medical soap products is preferred due to the negative or hazardous consequences of these chemicals.Recent years have seen a rise in the use of plant-based natural products as a synthetic ingredient to enhance the essential biological qualities of medicinal soap [6][7][8][9].One of the main medicinal plants that originated in the tropics and subtropics is Indian Sorrel.It can used as a traditional medicine in systems of traditional medicine like Ayurveda, Unani, and Siddha [10][11][12][13].Oxalidaceae can cure liver problems, skin conditions, and urinary tract issues in conventional medicine.Indian Sorrel contains fatty acids, including stearic, palmitic, oleic, and linolenic acids, all of which have different pharmacological effects [14][15][16].Vitamin E, which has biological action as an antioxidant, is present in the methanol extract of O. corniculata [17][18][19].The entire O. corniculata has been used to isolate the sitosterol components betulin, p-hydroxybenzoic acid, ethyl gallate, methoxy flavones, etc. [20][21][22][23].Due to lack of proper knowledge and experimental evidence a huge amount of agricultural resources are mismanaged in the form of agro wastes despite possessing beneficial health effects [24,25].
In our present study, the scavenging activity and antimicrobial efficiency of leaves of Oxalis corniculata Linn.was evaluated, also a therapeutic soap formulation was created.

Plant extract preparation and therapeutic soap formulation
The whole plant of Oxalis corniculata Linn.was obtained from the campus of Adamas University located in Barasat, West Bengal, India (Coordinates 22.740868681325516, 88.45751085452098) as shown in Fig. 1.The plant Indian Sorrel (Oxalis corniculata Linn) were authenticated by the taxonomist of the Botanical Survey of India, Kolkata (CNH/Tech.II/2024/24).Samples were allowed to dry at 30 °C for 24 h followed by homogenizing using a mortar and pestle to prepare the extract.Two grams of powdered plant (Oxalis corniculata Linn) was used for extraction using 10 ml of three different solvents including 80 % ethanol (Et), 80 % methanol (Mt) and 100 % distilled water (dH 2 O) respectively.The extracts were dried to powder using a rotary vacuum evaporator (Superfit ROTAVAP PBU-6).Ten percent w/v solutions of each of powder were used as samples.The necessary chemicals were procured from different reputed companies like Folin-Ciocalteu reagent (P20571), ascorbic acid (A33117) was purchased from NICE, DPPH (29128) and ABTS (28042) were obtained from SRL.
The market available soap base was heated in the microwave for melting and collected in a beaker.Freshly prepared dried powder 10 % w/v of ethanolic extract of O. corniculata Linn.was poured onto the mould and allowed to cool for 24 h.The mould was removed after the soap was formed as shown in Fig. 2a.The whole experimental design from plant extract preparation to soap formulation is depicted in a flowchart below (Fig. 2b).

Determination of phenol content and flavonoid content
The total phenolic content (TPC) of Oxalis corniculata Linn.plant extract in different solvents and formulated soap were determined with the help of colorimetric Folin-Ciocalteu reagent using gallic acid as standard.A reaction mixture containing 100 µl of plant extract, 1000 µl of both Folin-Ciocalteu reagent (diluted 20 times) and sodium carbonate (7 %) was incubated in the dark for 1 h 30 min and absorbance at 760 nm was taken with a spectrophotometer (Hitachi, U-2910) and the results were reported as gallic acid equivalent in milligrams GAE per gram Dry Weight (DW).The total flavonoid content (TFC) of Oxalis corniculata Linn. in different solvent extracts and formulated soap were measured by a spectrophotometric method.Ten millilitres of total reaction mix including 1 ml of sample extract and 4 ml of distilled water was incubated for 5 min and mixed with 0.3 ml of 5 % sodium nitrite and 0.3 ml of 10 % aluminium chloride to incubate for 6 mins.Then 1 ml sodium hydroxide (1 M) was poured into the reaction mix, making a final volume of 10 ml.After 1 min, the absorbance was measured at 510 nm and results were shown as Quercetin equivalent (QE) of Dry Weight (DW) [26][27][28].

Reducing power assay and total antioxidant assay
Measurement of antioxidant potential of Oxalis corniculata Linn.plant extract in different solvents and formulated soap were carried out by estimating the reduction of ferric iron to ferrous iron (ferric reducing power/ FRAP assay).A phosphomolybdenum method was also carried out to evaluate total antioxidant capacity (TAC) of Oxalis corniculata Linn. in different solvent extracts and formulated soap [29][30][31].

ABTS assay
The antioxidant potential of Oxalis corniculata Linn. in different solvent extracts and formulated soap were determined by ABTS free radical scavenging assay.Briefly, two solutions of 7 mM 2,2′-azinobis-(3-ethylbenzothiaziline-6-sulfonate) and 2.45 mM of potassium persulfate were mixed in 1 : 1 ratio and kept in the dark up to 24-48 h.Two millilitres of ABTS•+ solution diluted with aqueous methanol in 1 : 25 ratio was added in an aliquot of ten times diluted 20 µl plant extract and incubated at 30 °C followed by reading the absorbance at 734 nm after 0, 10, and 20 min where ascorbic acid serves as standard [32][33][34].

DPPH radical scavenging method
DPPH radical scavenging potential of Oxalis corniculata Linn. in different solvent extracts and formulated soap were determined through a spectrophotometric method using ascorbic acid as a standard.A volume of 0.5 ml of plant extract was mixed properly with 2 ml of 1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) solution.The absorbance was taken at 517 nm after 1 h incubation at room temperature [34].

Agar well diffusion method
This method was used to measure the antibacterial activity of the Oxalis corniculata Linn. in different solvent extracts and formulated soap against two Gram-positive bacteria Staphylococcus aureus (ATCC25923-0360P) and Bacillus subtilis (ATCC11774-0269P), and two Gram-negative bacteria Escherichia coli (ATCC35218-0495P) and Salmonella typhi (ATCC14028-0363P).The bacterial strains were cultured in 20 ml nutrient broth medium and incubated for a day with 101 revolutions per minute (r.p.m.) (New Brunswick Scientific Excella E24 Incubator Shaker) to enhance their growth.The wells were made with a sterile cork borer after spreading the inoculum over the agar into which plant extracts were poured and incubated at 37 °C for 24 h to obtain zones of growth inhibition.Here, chloramphenicol was used as positive control [35,36].

Determination of minimum inhibitory concentration (mIC)
The antibacterial potential of the different decoctions of whole plant of Oxalis corniculata Linn.was evaluated by microbroth dilution assay in 96-well plates.The desired inoculum of bacterial strains were prepared overnight at 37 °C in Mueller-Hilton agar broth possessing 0.5 McFarland turbidity standard at 600 nm OD (optical density).Then 100 µl Mueller-Hilton agar was added in all the 12 wells of each row.In wells 4-12, 10 µl of varying concentrations (0.04-10 µg ml −1 ) of different decoctions of Oxalis corniculata Linn.were added.Additionally, each well received 5 µl of bacterial suspension except the first well which served as negative control.The microtitre plate was allowed to incubate at 37 °C for 24 h and growth was observed by visual inspection.The lowest concentration of the extract at which no visible growth was observed was recorded as the MIC.

Experimental design and statistical analysis
Every experiment was performed thrice.One-way ANOVA was used to determine the difference in mean values and represented in the form of mean±standard deviation using GraphPad Prism5 software Version 5.03 (San Diego, United States of America).After performing an ANOVA to determine the impact of various wavelengths and the percentage of solvent used on the physical qualities of the soap, the process was optimized using response surface technique.With two independent variables (factors), such as different wavelengths (X 1 ) and the percentage of solvent used (X 2 ), on the dependent variables (responses), absorbance (Y 1 ) was measured.Five centre points were used in thirteen trials.Table 1 lists the two factors together, along with their actual and coded values.The design used five replicates in the centre.
According to lack-of-fit tests, multiple correlation coefficients (R 2 ), sequential model sum of squares (SMSS), and lack-of-fit-tests, linear, quadratic, or cubic models for each response variable were chosen.Because there weren't enough points to support a cubic model, it was aliased.With this data, a quadratic model was selected as the most plausible explanation.The following quadratic regression formula was used to fit the experimental response functions of second order: Where X 1 is the different wavelength; X 2 is the percentage of solvent used; the fitted response value at the centre of the design, point (0,0), is β 0 ; the linear regression terms are β 1 and β 2 ; the quadratic regression terms are β 11 and β 22 ; and the cross product regression term is β 12 .The means for the scores obtained for each of the quality attributes of each product were independently calculated after three replications of each study.The software Design Expert Version 7.1.6(Stat-Ease, Inc., Minneapolis, USA) was used to perform statistical analysis of variance (ANOVA) and multiple regressions in order to fit the equation.Results included factors like predicted model coefficients, a goodness-of-fit test, and regression coefficients.

High-performance liquid chromatography (HPLC) analysis
Indian Sorrel (Oxalis corniculata Linn.) samples were analysed for the detection of bioactive compounds in a C18 reverse phase HPLC column system with dimensions 4.6 mm × 250 mm, particle size 5 µm.The mobile phase consists of 0.1 % trichloroacetic acid in HPLC grade H 2 O and 90 % acetonitrile in a constant gradient.A diluted extract was injected in a volume of 10 µl.The flow rate was 1 ml min −1 .Total run time was 15 min.The chromatograms were obtained at 254 nm for analysis of hesperidin, quercetin and gallic acid.The identification as well as quantitation of bioactive compound in sample extract were carried out by using the retention time, absorption spectra, peak areas of the respective standard and expressed as micrograms per millilitre (μg ml −1 ) [29,35].

Antibacterial activity assay
The 80 % methanolic and 80 % ethanolic decoctions of whole plant of Indian Sorrel portray significant antibacterial properties against four bacterial strains (Fig. 7).Here, chloramphenicol (C30) showed highest antibacterial activity by serving as a positive control.Among all extracts, the 80 % ethanolic decoctions showed highest zone of inhibition with diameter ranges from 14.650±0.141mm for Escherichia coli, 23 2).

minimum inhibitory concentration
The effectiveness of different decoctions of whole plant of Oxalis corniculata Linn. in different bacterial strains was evaluated by determining the minimum inhibitory concentration (MIC) (Fig. 8).Among all different decoctions tested, the 80 % ethanolic decoction always exhibits the strongest antimicrobial potential.The MIC of two Gram-negative bacterial strains, i.e.Escherichia coli and Salmonella typhimurium were 0.625 µg ml −1 and 5 µg ml −1 respectively.The MIC of Bacillus subtillis was 2.5 µg ml −1 and Staphylococcus aureus was 5 µg ml −1 .The 80 % methanolic decoctions of whole plant of Indian Sorrel was also effective against Escherichia coli, Salmonella typhimurium, Bacillus subtillis, and Staphylococcus aureus with MIC values of 0.08 µg ml −1 , 0.625 µg ml −1 , 0.08 µg ml −1 , and 0.08 µg ml −1 , respectively.The formulated soap exerts greater MIC values (0.3 µg ml −1 against both Escherichia coli and Bacillus subtillis, 0.625 µg ml −1 against Salmonella typhimurium and Staphylococcus aureus) as compared to the market soap.

Effects of different wavelengths and percent of solvent used on absorbance
ANOVA for the RSM quadratic model 80 % ethanolic and 80 % methanolic decoction was given in Tables 3 and 4 respectively.The regression coefficient value of 80 % ethanolic and 80 % methanolic decoction of this model were 0.75 and 0.70 respectively.More fitted data are shared by 80 % methanolic decoction in the response data used to fit the model.The combined impact of various wavelengths and the percentage of solvent used on absorbance is shown in Fig. 9.

Detection of bioactive compounds
In order to ascertain whether or not any bioactive compound was present in samples such as crude and product, we analysed the samples by HPLC.The peak at 1.371 min was identified as that of hesperidin.Sample crude and product also showed a peak at the same retention time, i.e. 1.479 and 1.480 respectively at 254 nm which corresponded to concentration of hesperidin 0.0933 mg ml −1 in crude Oxalis corniculata Linn.and 0.0542 mg ml −1 in OC soap as shown in figure.Similarly, a peak at 1.520 min was identified for quercetin.Sample crude and product showed a peak at 254 nm at the same retention time, i.e. 1.588 and 1.480 respectively as  the quercetin standard which corresponded to concentration of quercetin 0.1968 mg ml −1 in crude Oxalis corniculata Linn.and 0.1462 mg ml −1 in OC soap as shown in Figure .Likewise, the peak at 1.388 min was identified as that of gallic acid.Sample crude and product showed a peak at 254 nm at the same retention time, i.e. 1.364 and 1.361 respectively as the gallic acid standard which corresponded to concentration of gallic acid 0.0917 mg ml −1 in crude Oxalis corniculata Linn.and 0.0682 mg ml −1 in OC soap as shown in Fig. 10.

DISCuSSIon
In the past several decades, a huge number of reports have explored the antioxidant, cardioprotective, neuroprotective, antidiabetic, antihypertensive, anti-inflammatory, and anticarcinogenic potential of different bioactive compounds isolated from low-cost agricultural resources [26,37].For instance, apple peels, grape skins, avocado seeds, etc. possess approximately 15 % higher polyphenolic compounds as compared to the fruit pulp.However, due to lack of proper knowledge and experimental evidence a huge number of agricultural resources are mismanaged in the form of agro wastes despite possessing beneficial health effects.The herbaceous plant Oxalis corniculata Linn.(Oxalidaceae) is commonly used as an antibacterial, refrigerant, diaphoretic, and wound-healing agent in traditional medical practises such as Ayurveda, Unani, and Siddha [3,10].It is enriched with several bioactive compounds including essential fatty acids, vitamin C, carotenoids, flavonoids, phytosterol, phenol, tannins, etc. [20,38].
Although several studies have been conducted to explore the potential of the Indian Sorrel leaf extract, the scavenging activity as well as antimicrobial efficiency of Oxalis corniculata Linn.leaf extract in different decoctions were investigated in this study due to the importance and necessity of formulating a therapeutic soap.In the present study, two free radical scavenging activity assays (DPPH, ABTS) were used to ascertain the antioxidant potential.
The results obtained from these tests were almost consistent, i.e. the 80 % ethanolic extract exhibited maximum radical scavenging activity as well as antioxidant properties which may be due to its greater TPC and TFC.In one earlier study, it was reported that oral administration of ethanolic extract of Oxalis corniculata in Wistar rats attenuated the oxidative stress mediated hepatotoxic effects of thioacetamide.The antioxidant constituents such as flavonoids and vitamin C present in ethanolic extract of Oxalis corniculata were mainly responsible for offering this hepatoprotective activity [39].In this research, among the 80 % ethanolic, 80 % methanolic and 100 % distilled water decoctions of Oxalis corniculata Linn.plant extracts, the 80 % ethanolic decoction of Oxalis corniculata Linn.plant showed significantly higher bacterial growth inhibitory properties in agar well diffusion assays and MIC assays followed by the 80 % methanolic decoction against four different bacterial strains.This result was similar to a study conducted by [40] which revealed that leaves of Oxalis corniculata possess highest antibacterial potential due to its higher flavonoid and phenolic contents than the seeds [40,41].Another report [42], found that methanolic and ethanolic extracts of Oxalis corniculata Linn.plant showed significant antibacterial activity against phytopathogenic (e.g.Xanthomonas pathovars) as well as human pathogenic bacteria with the help of its phenolic constituents [42].Phytochemical analysis revealed that in ethanolic and methanolic extracts of Oxalis corniculata, the presence of phenolic compounds, flavonoids, carbohydrates, amino acids, proteins, volatile oil, citric acid, tartaric acid, fibres, and tannins are higher [42,43].The HPLC analysis performed in this study had confirmed the presence of hesperidin, quercetin and gallic acid in the crude sample and OC soap which are responsible for governing the antioxidant and antibacterial activities.Therefore, according to the results obtained in this research, the 80 % ethanolic extract of Oxalis corniculata Linn.was selected to be used to achieve good antioxidant and antibacterial activity in herbal soap formulation.
Presently, although there is a wide range of standard soaps are available in the market, however it has some drawbacks such as its preservatives content, petroleum based lathering agents such as synthetic fragrances, harsh dyes or colouring agents, etc. which over long-time usage may lead to harmful effects on the skin.In contrast to this, the organic soaps which are made up of natural ingredients, herbs or any sort Ayurvedic compound are cruelty-free and animal-friendly.The pH level of organic products is between 9 and 10 (much basic in nature).It means that it is gentler on your skin, and it makes the skin less prone to irritation.Organic soaps are loaded with glycerine, also known as glycerol, which is also a natural alcohol and water attractor that is found  in soaps.Beside from its cleansing properties, it is considered the best moisturizing agent for the human skin, and has been in use for centuries.Organic products use natural antibacterial agents such as essential oils (like peppermint, tea-tree, lavender, and lemongrass) for cleansing, moisturizing and adding fragrance.Therefore, nowadays organic soaps are gaining more preference over the markedly available soaps [44].These include soaps manufactured with dried leaves of basil, neem, acalypha, aloe vera, and hibiscus flowers by stream extraction process.In this study, an organic soap was formulated with Oxalis corniculata Linn.which offered significantly higher phenolic, flavonoid content, antioxidant and antibacterial potential in comparison to the market soap, which may be due to the enrichment of different beneficial bioactive compounds in the Oxalis corniculata Linn.extract.In one similar study done earlier, a bath soap formulated with different bioactive herbal plant extracts was compared with market soap.The herbal soap besides having good appearance, colour, and fragrance, was also found to possess good antioxidant and antibacterial properties due to its constituent bioactive components [45].A soap formulated with seed coat of Borassus flabellifer and rhizome of Curcuma zedoaria displayed good antimicrobial activity due to the bioactive compounds present in the herbal extract as determined by GC MS analysis [46].The liquid medicinal soap prepared from pomegranate (Punica granatum) leaf extracts retained both the actual antioxidant and antibacterial activity of the crude plant extract, whereas the solid soap possesses only the antioxidant activity [47].Utilization of this kind of antioxidant as well as antibacterial active soap in our daily lifestyle can improve the quality of our skin health and also counteract the rising skin disease death cases.

Fig. 2 .
Fig. 2. (a) Soap made with Oxalis corniculata Linn extract.(b) Flow chart representing the steps of herbal soap formulation from Oxalis corniculata Linn leaf extract.

Fig. 9 .
Fig. 9.The 3D RSM plots to visualize the interaction effects of the variables in (a) 80 % ethanol and (b) 80 % methanol extract.

Table 2 .
Table showing the zone of growth inhibition (in mm) by the Oxalis corniculata Linn in different solvent extracts and formulated soap against different bacterial strains.The values are shown as mean±SD (n=3) with *0.05≥P, **0.01≥P, ***0.001≥P.Here the inhibition of bacterial strains are compared with chloramphenicol C30